Molecular Determinants of Stereoselective Bupivacaine Block of hKv1.5 Channels

Abstract

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Structural Basis for Competition between Drug Binding and Kvβ1.3 Accessory Subunit-Induced N-Type Inactivation of Kv1.5 Channels

“…Homology models are based on the crystal structure of the KcsA channel (Doyle et al, 1998) the ability of Kv␤1.3 subunits to alter Kv1.5 channel gating. However, mutation of Leu510 was previously reported to decrease channel block by the local anesthetics quinidine and tetraethylammonium (Yeola et al, 1996;Franqueza et al, 1997;Caballero et al, 2002).…”

Molecular Basis for Kv1.5 Channel Block

“…However, with the exception of Thr-479, these residues were not previously reported to influence the pharmacology of Kv1.5. Based on a more limited mutagenesis approach, the S6 segment residues, Thr-507, Leu-510, and Val-514, were identified as potential sites of drug-channel interactions for quinidine, benzocaine, and bupivacaine (7,8,11). However, the Kv1.3/1.5 homology model predicts that these residues point away from the central cavity.…”

“…The T479S mutation is equivalent to the T441S mutation in Shaker channels, which reduced internal TEA affinity 10-fold (12). This residue is also important for binding of bupivacaine (8), benzocaine (11), and S0100176. Interestingly, the IC 50 of S0100176 was increased even more by mutation of Thr-480 to Ser.…”

“…The residues Thr-507, Leu-510, and Val-514 of the S6 segment (7,8) and Thr-479 near the selectivity filter (8,11) were identified as potential binding sites for these drugs. Previous studies have shown that blockers of Na ϩ , Ca 2ϩ , and K ϩ channels also bind to residues within the S6 segment and/or regions near the selectivity filter (12)(13)(14)(15)(16)(17)(18).…”

Binding Site of a Novel Kv1.5 Blocker: A “Foot in the Door” against Atrial Fibrillation

“…Quinidine, bupivacaine, and benzocaine interact with residues located near the pore helix (Thr479) and in the S6 domain (Thr507, Leu510, and Val514) of Kv1.5 (Snyders et al, 1992;Snyders and Yeola, 1995;Yeola et al, 1996;Franqueza et al, 1997;Caballero et al, 2002). More recently discovered Kv1.5 blockers, such as the anthranilic acid derivative S0100176, have also been shown to bind to residues located in the channel pore.…”